The goal of this proposal is to investigate the earliest changes during ischemia/reperfusion (I/R) injury of the heart. During the tenure of the present support, much of the research has focused on the biochemical and morphological changes during the first minutes of reoxygenation of cells (a/R) after an initial anoxic cellular event. Both the I/R heart model and isolated A/R cellular models show an initial burst of oxyradicals, along with two peaks of alkoxyl radicals, providing an experimental time-course, during which changes in lipids, proteins, enzymatic activities and morphological parameters can be sampled. The primary events producing the initial and secondary bursts of free radicals require further study. The "delocalization" of iron within the cell may participate in triggering oxyradical production and propagating the subsequent free radical injury. Thus, the proposed research plan addresses the hypothesis that during the anoxia/reoxygenation process iron is released from "appropriate" subcellular binding sites to other areas of the cell, where it is re-bound to "ectopic" sites to trigger localized injury of lipids and proteins. The novel biochemical endpoints to be employed include: measurements of alkoxyl radicals, lipid peroxides, protein/enzyme oxidation, analyses of free iron, subcellular localization of iron (X-ray microanalysis) and peroxidation products (Meridian ACAS microscope), and phospholipase-activation. The clinically relevant goals of the proposed research are to understand the role of free radicals in the reperfusion injury process and the mechanisms by which antioxidant agents, particularly those drugs that may have immediate applications in patient therapy, are producing their protective effects.